Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques.

BACKGROUND: The fight against COVID-19 requires mass vaccination strategies, and vaccines inducing durable cross-protective responses are still needed. Inactivated vaccines have proven lasting efficacy against many pathogens and good safety records. They contain multiple protein antigens that may improve response breadth and can be easily adapted every year to maintain preparedness for future seasonally emerging variants. METHODS: The vaccine dose was determined using ELISA and pseudoviral particle-based neutralization assay in the mice. The immunogenicity was assessed in the non-human primates with multiplex ELISA, neutralization assays, ELISpot and intracellular staining. The efficacy was demonstrated by viral quantification in fluids using RT-qPCR and respiratory tissue lesions evaluation. RESULTS: Here we report the immunogenicity and efficacy of VLA2001 in animal models. VLA2001 formulated with alum and the TLR9 agonist CpG 1018™ adjuvant generate a Th1-biased immune response and serum neutralizing antibodies in female BALB/c mice. In male cynomolgus macaques, two injections of VLA2001 are sufficient to induce specific and polyfunctional CD4+ T cell responses, predominantly Th1-biased, and high levels of antibodies neutralizing SARS-CoV-2 infection in cell culture. These antibodies also inhibit the binding of the Spike protein to human ACE2 receptor of several variants of concern most resistant to neutralization. After exposure to a high dose of homologous SARS-CoV-2, vaccinated groups exhibit significant levels of protection from viral replication in the upper and lower respiratory tracts and from lung tissue inflammation. CONCLUSIONS: We demonstrate that the VLA2001 adjuvanted vaccine is immunogenic both in mouse and NHP models and prevent cynomolgus macaques from the viruses responsible of COVID-19.

Mass vaccination in response to the COVID-19 pandemic has substantially reduced the number of severe cases and hospitalizations. As the virus continues to evolve and give rise to new variants that cause local outbreaks, there is a need to develop new vaccine candidates capable of stopping the viral transmission. In this study, we explore the immune responses induced by the vaccine candidate VLA2001 in animal models. We highlight the vaccine's ability to induce an immune response capable of blocking the virus and eliminating infected cells. We show that it can protect the host from developing severe disease.

Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates.

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

Impact of a PMMA tube on performances of a Vereos PET/CT system adapted for BSL-3 environment according to the NEMA NU2-2012 standard.

INTRODUCTION: A Vereos PET/CT device was adapted to be compatible with the experimentation in large animals within BSL-3 environment. The aim of this study was to investigate the impact of this modification on the performance according to NEMA NU2-2012 standard. METHODS: Spatial resolution, sensitivity, count rate performance, accuracies of corrections and image quality were assessed using the NEMA NU2-2012 standards before and after installation of a transparent poly-methyl methacrylate tube of 8 mm thickness, 680 mm diameter and 2800 mm long inside the tunnel of the system. In addition, CT performance tests were performed according to manufacturer standard procedure. RESULTS: Although the presence of the tube led to a slight decrease in sensitivity, performance measurements were in accordance with manufacturer preconisation ranges and comparable to previous performance published data. CONCLUSION: Modifications of Vereos PET/CT system allowing its use in BSL-3 conditions did not affect significantly its performance according to NEMA NU2-2012 standard. KEY POINTS: Question. Does a BSL-3 compatible modification alter Philips Vereos PET/CT performances according to NEMA NU2-2012 standards? Pertinent findings. Our Vereos PET/CT system was modified by a wall separating BSL-1 and BSL-3 sides and an 8 mm thickness PMMA tube inserted into the bore of the camera in order to extend the BSL-3 containment along the bed movement. The performances of our modified system according to NEMA NU2-2012 standards were not significantly impacted by the modifications and were in accordance with the values prescribed by the manufacturer. Implications for patients care. Our clinical PET/CT device was modified for human infectious diseases studies in Non-Human Primates. This unusual set up may then provide truly transposable data from preclinical studies into clinical application in infected patients.

Computed tomography and [18F]-FDG PET imaging provide additional readouts for COVID-19 pathogenesis and therapies evaluation in non-human primates.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [18F]-FDG positron emission tomography (PET) to monitor the early phase of the disease in a large cohort (n = 76) of SARS-CoV-2 infected macaques. Following infection, animals showed mild COVID-19 symptoms including typical lung lesions. CT scores at the acute phase reflect the heterogeneity of lung burden following infection. Moreover, [18F]-FDG PET revealed that FDG uptake was significantly higher in the lungs, nasal cavities, lung-draining lymph nodes, and spleen of NHPs by 5 days postinfection compared to pre-infection levels, indicating early local inflammation. The comparison of CT and PET data from previous COVID-19 treatments or vaccines we tested in NHP, to this large cohort of untreated animals demonstrated the value of in vivo imaging in preclinical trials.

Intranasal inoculation with Bordetella pertussis confers protection without inducing classical whooping cough in baboons.

BACKGROUND: The resurgence of whooping cough in many countries highlights the crucial need for a better understanding of the pathogenesis of respiratory infection by Bordetella pertussis. Exposure of baboons to B. pertussis by the intranasal and intra-tracheal routes is a recently described preclinical model that reproduces both B. pertussis infection of humans and whooping cough disease. Here, we tested both intranasal and intranasal+intra-tracheal exposure routes and assessed their impact on disease development and immunity. METHODS: Young baboons were intranasally exposed to the B1917 clinical isolate, representative of circulating strains in Europe, or its green-fluorescent protein expressing derivative. Animals were followed for pertussis symptoms and bacterial colonization and by in vivo probe-based confocal laser endomicroscopy (pCLE) imaging. Sero-conversion and protection against subsequent infection were then evaluated. RESULTS: Seroconversion and bacterial colonization of both the nasopharynx and trachea was observed in baboons exposed to B. pertussis by the intranasal route only, and also in those animals challenged by both the intranasal and intra-tracheal routes together. However, baboons exposed solely by the intranasal route developed only mild clinical symptoms, with no paroxysmal cough. These animals were protected against re-infection by B. pertussis. CONCLUSIONS: Intranasal exposure of baboons to B. pertussis does not induce disease but elicits immune mechanisms that protect them from subsequent exposure to the bacteria. These findings suggest that the intranasal route of inoculation in this non-human primate model could be used in the pre-clinical evaluation of nasal candidate vaccines against pertussis.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

Effective treatments against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Monoclonal antibodies have shown promising results in patients. Here, we evaluate the in vivo prophylactic and therapeutic effect of COVA1-18, a neutralizing antibody highly potent against the B.1.1.7 isolate. In both prophylactic and therapeutic settings, SARS-CoV-2 remains undetectable in the lungs of treated hACE2 mice. Therapeutic treatment also causes a reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg-1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 shows very strong antiviral activity in the upper respiratory compartments. Using a mathematical model, we estimate that COVA1-18 reduces viral infectivity by more than 95% in these compartments, preventing lymphopenia and extensive lung lesions. Our findings demonstrate that COVA1-18 has a strong antiviral activity in three preclinical models and could be a valuable candidate for further clinical evaluation.

Targeting SARS-CoV-2 receptor-binding domain to cells expressing CD40 improves protection to infection in convalescent macaques.

Achieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.

An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates.

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.

Predictive Markers of Immunogenicity and Efficacy for Human Vaccines.

Vaccines represent one of the major advances of modern medicine. Despite the many successes of vaccination, continuous efforts to design new vaccines are needed to fight "old" pandemics, such as tuberculosis and malaria, as well as emerging pathogens, such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccination aims at reaching sterilizing immunity, however assessing vaccine efficacy is still challenging and underscores the need for a better understanding of immune protective responses. Identifying reliable predictive markers of immunogenicity can help to select and develop promising vaccine candidates during early preclinical studies and can lead to improved, personalized, vaccination strategies. A systems biology approach is increasingly being adopted to address these major challenges using multiple high-dimensional technologies combined with in silico models. Although the goal is to develop predictive models of vaccine efficacy in humans, applying this approach to animal models empowers basic and translational vaccine research. In this review, we provide an overview of vaccine immune signatures in preclinical models, as well as in target human populations. We also discuss high-throughput technologies used to probe vaccine-induced responses, along with data analysis and computational methodologies applied to the predictive modeling of vaccine efficacy.

Leukocytospermia induces intraepithelial recruitment of dendritic cells and increases SIV replication in colorectal tissue explants.

Mucosal exposure to infected semen accounts for the majority of HIV-1 transmission events, with rectal intercourse being the route with the highest estimated risk of transmission. Yet, the impact of semen inflammation on colorectal HIV-1 transmission has never been addressed. Here we use cynomolgus macaques colorectal tissue explants to explore the effect of leukocytospermia, indicative of male genital tract inflammation, on SIVmac251 infection. We show that leukocytospermic seminal plasma (LSP) has significantly higher concentration of a number of pro-inflammatory molecules compared to normal seminal plasma (NSP). In virus-exposed explants, LSP enhance SIV infection more efficiently than NSP, being the increased viral replication linked to the level of inflammatory and immunomodulatory cytokines. Moreover, LSP induce leukocyte accumulation on the apical side of the colorectal lamina propria and the recruitment of a higher number of intraepithelial dendritic cells than with NSP. These results suggest that the outcome of mucosal HIV-1 infection is influenced by the inflammatory state of the semen donor, and provide further insights into mucosal SIV/HIV-1 pathogenesis.

Visualization of HIV-1 reservoir: an imaging perspective.

PURPOSE OF REVIEW: The persistence of HIV-1-infected cells, despite the introduction of the combinatorial antiretroviral therapy, is a major obstacle to HIV-1 eradication. Understanding the nature of HIV reservoir will lead to novel therapeutic approaches for the functional cure or eradication of the virus. In this review, we will update the recent development in imaging applications toward HIV-1/simian immunodeficiency virus (SIV) viral reservoirs research and highlight some of their limitations. RECENT FINDINGS: CD4 T cells are the primary target of HIV-1/SIV and the predominant site for productive and latent reservoirs. This viral reservoir preferentially resides in lymphoid compartments that are difficult to access, which renders sampling and measurements problematical and a hurdle for understanding HIV-1 pathogenicity. Novel noninvasive technologies are needed to circumvent this and urgently help to find a cure for HIV-1. Recent technological advancements have had a significant impact on the development of imaging methodologies allowing the visualization of relevant biomarkers with high resolution and analytical capacity. Such methodologies have provided insights into our understanding of cellular and molecular interactions in health and disease. SUMMARY: Imaging of the HIV-1 reservoir can provide significant insights for the nature (cell types), spatial distribution, and the role of the tissue microenvironment for its in vivo dynamics and potentially lead to novel targets for the virus elimination.

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Innate Molecular and Cellular Signature in the Skin Preceding Long-Lasting T Cell Responses after Electroporated DNA Vaccination.

DNA vaccines delivered with electroporation (EP) have shown promising results in preclinical models and are evaluated in clinical trials. In this study, we aim to characterize early mechanisms occurring in the skin after intradermal injection and EP of the auxoGTUmultiSIV DNA vaccine in nonhuman primates. First, we show that EP acts as an adjuvant by enhancing local inflammation, notably via granulocytes, monocytes/macrophages, and CD1aint-expressing cell recruitment. EP also induced Langerhans cell maturation, illustrated by CD86, CD83, and HLA-DR upregulation and their migration out of the epidermis. Second, we demonstrate the crucial role of the DNA vaccine in soluble factors release, such as MCP-1 or IL-15. Transcriptomic analysis showed that EP played a major role in gene expression changes postvaccination. However, the DNA vaccine is required to strongly upregulate several genes involved in inflammatory responses (e.g., Saa4), cell migration (e.g., Ccl3, Ccl5, or Cxcl10), APC activation (e.g., Cd86), and IFN-inducible genes (e.g., Ifit3, Ifit5, Irf7, Isg15, orMx1), illustrating an antiviral response signature. Also, AIM-2, a cytosolic DNA sensor, appeared to be strongly upregulated only in the presence of the DNA vaccine and trends to positively correlate with several IFN-inducible genes, suggesting the potential role of AIM-2 in vaccine sensing and the subsequent innate response activation leading to strong adaptive T cell responses. Overall, these results demonstrate that a combined stimulation of the immune response, in which EP and the auxoGTUmultiSIV vaccine triggered different components of the innate immunity, led to strong and persistent cellular recall responses.

Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques.

BACKGROUND: Autoimmune demyelinating diseases (ADD) are a major cause of neurological disability due to autoreactive cellular and humoral immune responses against brain antigens. A cure for chronic ADD could be obtained by appropriate immunomodulation. METHODS: We implemented a preclinical scheme to foster immune tolerance to myelin oligodendrocyte glycoprotein (MOG), in a cynomolgus-macaque model of experimental autoimmune encephalomyelitis (EAE), in which administration of recombinant human MOG (rhMOG) elicits brain inflammation mediated by MOG-autoreactive CD4+ lymphocytes and anti-MOG IgG. For immunotherapy, we used a recombinant antibody (Ab) directed against the dendritic cell-asialoglycoprotein receptor (DC-ASGPR) fused either to MOG or a control antigen PSA (prostate-specific antigen). FINDINGS: rhMOG and the anti-DC-ASGPR-MOG were respectively detected in CD1a+ DCs or CD163+ cells in the skin of macaques. Intradermal administration of anti-DC-ASGPR-MOG, but not control anti-DC-ASGPR-PSA, was protective against EAE. The treatment prevented the CD4+ T cell activation and proinflammatory cytokine production observed in controls. Moreover, the administration of anti-DC-ASGPR-MOG induced MOG-specific CD4+CD25+FOXP3+CD39+ regulatory lymphocytes and favoured an upsurge in systemic TGFß and IL-8 upon rhMOG re-administration in vivo. INTERPRETATION: We show that the delivery of an anti-DC-ASGPR-MOG allows antigen-specific adaptive immune modulation to prevent the breach of immune tolerance to MOG. Our findings pave the way for therapeutic vaccines for long-lasting remission to grave encephalomyelitis with identified autoantigens, such as ADD associated with anti-MOG autoantibodies. FUND: Work supported by the French ANR (ANR-11-INBS-0008 and ANR-10-EQPX-02-01), NIH (NIH 1 R01 AI 105066), the Baylor Scott and White Healthcare System funding and Roche Research Collaborative grants.

The Frequencies of Immunosuppressive Cells in Adipose Tissue Differ in Human, Non-human Primate, and Mouse Models.

Although the metabolic properties of white adipose tissue have been extensively characterized, the tissue's immune properties are now attracting renewed interest. Early experiments in a mouse model suggested that white adipose tissue contains a high density of regulatory T cells (Tregs), and so it was assumed that all adipose tissue has an immunosuppressive profile-even though the investigation was limited to visceral body fat in relatively old male mice. This observation was also corroborated by high frequencies of other cell subsets with immunoregulatory properties, such as anti-inflammatory M2 macrophages, and regulatory B cells. Many studies have since evidenced the persistence of pathogens (trypanosomes, Mycobacterium tuberculosis, HIV, etc.) in adipose tissue. However, a recent report identified adipose tissue as a reservoir of memory T cells capable of protecting animals upon rechallenge. The immune potential of lean adipose tissue thus remains to be further investigated. Here, we compared the relative proportions of immune cells (and Tregs in particular) in lean adipose tissue collected from humans, a non-human primate (the cynomolgus macaque), and three mouse models. We demonstrated that the proportion of Foxp3+ Tregs in visceral adipose tissue was low in all models other than the C57Bl/6 mouse. These low values were not linked to correspondingly low proportions of effector cells because T lymphocytes (a main target of Treg suppression) were more frequent in cynomolgus macaques than in C57Bl/6 mice and (to a lesser extent) humans. In contrast, the proportions of macrophages and B cells were lower in cynomolgus macaques than in C57Bl/6 mice. We also observed a higher proportion of CD34+CD45- cells (which predominantly correspond to mesenchymal stem cells) in C57Bl/6 mouse and cynomolgus macaques than in humans and both for subcutaneous and visceral adipose tissues. Lastly, a microscopy analysis confirmed predominant proportion of adipocytes within adipose tissue, and highlighted a marked difference in adipocyte size among the three species studied. In conclusion, our study of lean, middle-aged, male individuals showed that the immune compartment of adipose tissue differed markedly in humans vs. mice, and suggesting the presence of a more inflammatory steady-state profile in humans than mice.

In vivo imaging of bacterial colonization of the lower respiratory tract in a baboon model of Bordetella pertussis infection and transmission.

Recent whooping cough (pertussis) outbreaks in many countries highlight the crucial need for a better understanding of the pathogenesis of Bordetella pertussis infection of the respiratory tract. The baboon is a recently described preclinical model for the study of B. pertussis infection and may be ideal for the evaluation of new pertussis vaccines. However, many pathophysiological aspects, including bacterial localization and interactions, have yet to be described in this model. Here, we used a baboon model of infection with a fluorescent GFP-expressing B. pertussis strain, derived from European clinical isolate B1917. Juvenile baboons were used to evaluate susceptibility to infection and transmission. Non-invasive in vivo imaging procedures, using probe-based confocal endomicroscopy coupled with bronchoscopy, were developed to track fluorescent bacterial localization and cellular interactions with host cells in the lower respiratory tract of infected animals. All B1917-GFP-challenged animals developed classical pertussis symptoms, including paroxysmal cough, nasopharyngeal colonization, and leukocytosis. In vivo co-localization with antigen presenting cells and progressive bacterial colonization of the lower airways were also assessed by imaging during the first weeks of infection. Our results demonstrate that in vivo imaging can be used to assess bacterial colonization and to point out interactions in a baboon model of pertussis.

Molecular and Cellular Dynamics in the Skin, the Lymph Nodes, and the Blood of the Immune Response to Intradermal Injection of Modified Vaccinia Ankara Vaccine.

New vaccine design approaches would be greatly facilitated by a better understanding of the early systemic changes, and those that occur at the site of injection, responsible for the installation of a durable and oriented protective response. We performed a detailed characterization of very early infection and host response events following the intradermal administration of the modified vaccinia virus Ankara as a live attenuated vaccine model in non-human primates. Integrated analysis of the data obtained from in vivo imaging, histology, flow cytometry, multiplex cytokine, and transcriptomic analysis using tools derived from systems biology, such as co-expression networks, showed a strong early local and systemic inflammatory response that peaked at 24 h, which was then progressively replaced by an adaptive response during the installation of the host response to the vaccine. Granulocytes, macrophages, and monocytoid cells were massively recruited during the local innate response in association with local productions of GM-CSF, IL-1ß, MIP1α, MIP1ß, and TNFα. We also observed a rapid and transient granulocyte recruitment and the release of IL-6 and IL-1RA, followed by a persistent phase involving inflammatory monocytes. This systemic inflammation was confirmed by molecular signatures, such as upregulations of IL-6 and TNF pathways and acute phase response signaling. Such comprehensive approaches improve our understanding of the spatiotemporal orchestration of vaccine-elicited immune response, in a live-attenuated vaccine model, and thus contribute to rational vaccine development.

Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

Purpose: We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Procedures: Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). Results: The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. Conclusions: This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.